Leucine analogs were tested in the Krebs II
ascites cell-free translation system for the ability to inhibit preprotein cleavage by replacing
leucine in nascent chains of bovine
preprolactin, rat
preprolactin, human placental prelactogen (pre-hPL), and pre-alpha subunit of
human chorionic gonadotropin (alpha-hCG). In the absence of analog,
ascites microsomal membranes cleaved these preproteins to their mature forms and sequestered the processed products. Also, two
asparagine residues in alpha-hCG were glycosylated. When 4 mM beta-DL-hydroxyleucine was added to the lysate instead of
L-leucine, cotranslational processing and sequestration of both species of
preprolactin and pre-hPL were inhibited. Sequential Edman degradation confirmed that pre-hPL was not cleaved. The inhibition of processing by
beta-hydroxyleucine resulted from its incorporation into
protein. This was shown by reversal of the effect by addition of
leucine and by inhibition of [(3)H]
leucine incorporation into
protein. Of significance, the processing of pre-alpha-hCG was less sensitive to
beta-hydroxyleucine because its prepeptide contains only four scattered
leucine residues, whereas the presegments of hPL and the prolactins contain six to eight clustered
leucine residues. These experiments demonstrate that translocation and processing of secretory
proteins require structural features determined by the primary amino acid sequence.